The long-term objective of these studies is to investigate the mechanisms that regulate the degradation of extracellular matrix (ECM) in cartilage during development and arthritis. The matrix metalloproteinases (MMPs) are the major group of degradative enzymes in cartilage. These are inhibited by specific inhibitors, the Tissue Inhibitor of Metalloproteinases (TIMPs). TIMP-3 is a novel TIMP that is highly expressed in cartilage, and is unique in being a component of the ECM. The focus of this proposal is to examine the biological role of TIMP-3 during development, with particular emphasis on skeletal development, and to understand the biochemical basis for TIMP-3 interactions with ECM. This knowledge is critical for understanding cartilage destruction in arthritis and for developing new therapeutic strategies for arthritic diseases. To answer the first research question to be raised (i.e. whether cartilage absolutely deficient in TIMP-3 will be more susceptible to MMP degradation), the developmental consequences of abrogation of TIMP-3 will be studied in transgenic mice generated by targeted inactivation of TIMP-3 alleles. The structure of the TIMP-3 gene has been determined and will facilitate the rapid generation of targeting constructs for homologous recombination in embryonic stem cells and transgenic mice. Since skeletal development is characterized by high ECM turnover in cartilage, this offers a dynamic model system for determining the inhibitory role of TIMP-3. To assess the second question (i.e. what regions of TIMP-3 binds ECM, and which are the TIMP-3 ligand(s) in cartilage ECM), full length and truncated TIMP-3 proteins will be produced as soluble secreted fusion proteins with placental alkaline phosphatase (PLAP) as the fusion partner. These will be used as versatile probes to determine the ECM binding domain of TIMP-3 by transient transfections of fibroblasts and chondrocytes using PLAP enzyme activity or specific PLAP antibodies as a marker. PLAP- TIMP-3 fusion proteins, TIMP-3 synthetic peptides or anti-TIMP-3 antibodies will be used as probes in genetic or immunologic strategies designed to identify and characterize the ligand(s) of TIMP-3.